1. Field of the Invention
The present invention relates to the production of peptides in the milk of transgenic mammals, for example non-human placental mammals.
2. Related Art
Polymers of amino acids concatenated via their amino and carboxyl groups form the basis for a variety of important biological compounds. Polymers of 3 to 100 amino acids are generally known as peptides, whilst larger polymers are known as proteins. This distinction is purely arbitrary, and polymers of up to about 110 amino acids can still be considered as “peptides”. Thus, the term “peptide” as used herein refers to amino acid polymers of 3 to 110 amino acids. Peptides as defined herein may be biologically active without requiring any further modification, or may form the building blocks for larger complex molecules by chemical modification into larger structures or by modification such as glycosylation. The term “peptide” is used herein to include biologically active and inactive polymers, which may or may not have undergone further modification.
Peptides have a number of commercial applications, including use as medicaments, nutritional additives and research tools. For this reason, economic, large scale production of peptides is desirable. Direct chemical synthesis of peptides is expensive due to the high cost of reagents and the degree of purification needed to remove failed sequences. Microbial synthesis by recombinant DNA technology is an alternative, but not always appropriate for peptide production due to difficulties in extraction and purification from microbial cells, and the absence of microbial enzymes to perform necessary post-translational modification. Heterologous proteins may be produced in stably transfected mammalian host cell lines, many of which are commercially available today. However, concern remains that these cell lines are derived from tumours of various types.
As an alternative to the above methods, the production of proteins in the milk of transgenic sheep is possible, as illustrated in WO-A-8800239 and WO-A-9005188. The production of proteins in the milk of transgenic animals has the advantage that large volumes of milk containing the desired protein can be harvested using simple and environmentally safe technology. The use of living organisms to produce proteins means that all the material produced will be identical to the natural product. In terms of amino acid structures, this means that only L isomers will be produced. Also, the number of wrong sequences will be minimised due to the high fidelity of biological synthesis compared to synthetic routes.
Further, the use of a biological process for the production of the proteins ensures that only biologically safe materials are produced, in contrast to chemical methods where side reactions may produce toxic materials, which can only be removed at additional cost. The use of a biological process also enables some reactions, which are difficult to perform in good yield by chemical means, to be efficiently carried out. For example, carboxy terminal amidation of a peptide can be essential for biological activity or for the prolongation of in vivo half-life, and is carried out by a specific enzyme which recognises and modifies proteins having a glycine residue at the carboxy terminus (Eipper B. A et al., (1993) Protein Science 2 489–497). Therefore, suitably designed proteins produced by means of a transgenic animal will be specifically amidated prior to secretion. The amidation of proteins is only one of a number of post-translational modifications which can be carried out by the biosynthetic pathways in the mammary gland and harnessed for the synthesis of biologically active proteins. Other post-translational modifications include disulphide bridge formation, phosphorylation and γ-carboxylation of glutamic acid residues and the addition of O- and N-linked glycosylation (Wold, F. Ann. Rev. Biochem. 50 783–814).
The technology for the production of large proteins, as opposed to shorter peptides, in large quantities in the milk of transgenic sheep has been well established. For example, the human protease inhibitor, α1-antitrypsin has been produced in the milk of transgenic sheep in excess of thirty grams of protein per liter (Wright, G. et al., (1991) Bio/Technology, 9 77–84). It is expected that the same technology can be applied to the production of proteins in cattle, which can produce up to 10,000 liters of milk per lactation.
There are a number of difficulties relating to the secretion of short peptides in mammalian systems due to the nature of the secretory process. Proteins destined for secretion are directed into the endoplasmic recticulum, which forms the first stage of the constitutive secretory pathway, by a short pro-sequence, usually of at least twenty amino acids. The messenger RNA encoding a protein destined for secretion is translated by a ribosome which is initially free in the cytoplasm of the producing cell. However, as the end of the newly synthesised protein emerges from the large ribosome complex, the secretory leader sequence is bound to a ‘signal recognition protein’ (SRP). The act of binding has two effects. First, it causes the translation and protein synthetic machinery of the ribosome to ‘pause’ and secondly, it promotes the docking of the ribosome to the surface of the ER. This docking then re-starts translation and the protein destined for secretion is then synthesised through the ER membrane into the inner compartment. During the course of this second synthetic phase, the secretory leader sequence is cleaved off and the protein is folded appropriately. Then, after removal of the secretory leader sequence, and any secondary sequence related to correct folding, by proteolysis, and any other necessary modifications (primary glycosylation events, gamma carboxylation, etc) the protein moves on through the secretory pathway.
The fundamental problem with the secretion of peptides from mammalian systems is the requirement for a secretory leader sequence, the binding of the signal recognition peptide and the geometry of the ribosomal complex. Simple experiments have shown that if ribosomes which are actively translating proteins are treated with a powerful and non-specific protease, which can degrade all exposed proteins, then sequences of polypeptide about forty amino acids in length are protected. This implies that this sequence is buried within the large ribosomal complex and that only longer sequences capable of binding the SRP will be competent to enter the ER secretory pathway.
The requirement for a minimum peptide length was confirmed by studies which truncated normally secreted proteins such as lysozyme (Ibramimi et al (1986) Eur. J. Biochem 155(3) 571–6) and insulin (Okun et al (1990) J. Biol Chem. 265(13) 7478–84). Shorter versions of lysozyme, which still contained the secretory leader sequence, of 102 and 74 amino acids, were still capable of binding the SRP (as demonstrated by the ability of added SRP to ‘pause’ translation in a cell-free system) but a 52 amino acid truncation could not. Also, studies on the secretion of truncated insulin confirmed that not only did short peptides not ‘reach’ the SRP but were also secreted with low efficiency. Therefore, due to the basic mechanism of secretion it is evident that very short peptides cannot enter the secretory pathway. It is also apparent that even if peptides are long enough, with the addition of the secretory leader sequence, to engage the SRP, efficient secretion is unlikely due to a preference for amino acid sequences in excess of perhaps 100 amino acids (Okun et al (1990) J. Biol Chem. 265(13) 7478–84). This preference is reflected by the general size of secreted proteins which are normally at least 120 or more amino acids in length. Secretion of peptides shorter than 100 amino acids normally occurs via an entirely different mechanism where peptides are generated by the proteolytic cleavage of larger precurser proteins, sequestered in specialised vesicles within a cell and stored until needed. In this case secretion occurs in response to a specific signal which promotes fusion of the vesicle with the plasma membrane of the cell with concomitant release of the peptide into the external medium.
Thus, the basic mechanism by which proteins are secreted, involving ER docking mediated by the SRP, precludes the secretion of very short peptides, of less than perhaps 40 amino acids, and severely decreases the efficiency of peptides less than 100 amino acids long. In the absence of a fusion partner, to direct peptides to the secretory pathway, peptides of less than 100 amino acids long are naturally secreted by a completely different vesicle-based mechanism that only operates at high capacity in specialised tissues such as neurones. This pathway does not represent a viable alternative for making peptides in mammary tissue.
A second reason for expressing peptides as fusion proteins in milk is that it is easier to purify a fusion protein from milk, which is a complex biological fluid containing fats, sugars and proteins as well as peptides and proteolytic fragments, than to purify the free peptide. If the properties of the fusion partner dominate those of the peptide, it is likely that at least the initial purification steps will be common to processes for different peptides and thereby reduce development costs for a number of peptides. Regarding purification, the use of a peptide fusion is also beneficial in that two different recovery modalities can be employed: one for the fusion protein and then, after cleavage, one for the peptide. This approach is expected to yield a more pure product, or require fewer stages to achieve higher purity, because peptide impurities will be reduced during the purification of the fusion and protein impurities during the purification of the peptide.
The third advantage of expressing a peptide in milk as a fusion rather than as the peptide is that the biological properties of the pepide are likely to be masked and therefore not interfere with the physiology of the host animal. This has been demonstrated for calcitonin where it was shown that the alpha lactalbumin fusion protein was inactive in an in vivo assay designed to measure the depression of plasma calcium levels in the rat in response to an injection. This is in contrast to the cleaved and purified calcitonin which did exhibit biological activity (WO95/27782 and McKee C. et al (1998) Nat. Biotechnol. 
The expression of heterologous proteins in mature or fused form in the milk of a transgenic female animal is also described in WO92/22644. This application discloses fusing a peptide gene sequence into a HINDIII restriction enzyme site in the coding sequence of the WAP gene, in order to express the peptide in milk. This fused gene construct merely serves to target the peptide expression to milk, but does not result in the expression of a fusion protein in milk, and thus is likely to suffer from the above mentioned problems of the art.
WO 95/27782 describes processes for the production of peptides in the milk of transgenic animals based on expressing the peptide linked to a “fusion partner protein”. The fusion protein can be isolated from the milk and subsequently cleaved to release the desired peptide. In a preferred embodiment the use of human α-lactalbumin as a fusion partner protein linked to calcitonin as the desired peptide, is described. Human α-lactalbumin is a small, natural milk protein capable of terminal extension, thus satisfying some criteria of a fusion partner protein. However, it has demonstrated that human α-lactalbumin fusion constructs are expressed in the milk of rabbits at only 2.1 mg/ml (PCT/GB95/00769; McKee C. et al (1998) Nat. Biotechnol. 16(7) 647–651), a low yield compared to the yield of non-fusion α1-antitrypsin at 30 grams per liter. It is to this problem of low expression of such fusion proteins that the present invention is addressed.